HDAC8 Catalyzes the Hydrolysis of Long Chain Fatty Acyl Lysine

Abstract

The histone deacetylase (HDAC) family regulates many biological pathways through the deacetylation of lysine residues on histone and nonhistone proteins. Mammals have 18 HDACs that are classified into four classes. Class I, II, and IV are zinc-dependent, while class III is nicotinamide adenine dinucleotide (NAD⁺)-dependent lysine deacetylase or sirtuins. HDAC8, a class I HDAC family member, has been shown to have low deacetylation activity compared to other HDACs in vitro. Recent studies showed that several sirtuins, with low deacetylase activities, can actually hydrolyze other acyl lysine modifications more efficiently. Inspired by this, we tested the activity of HDAC8 using a variety of different acyl lysine peptides. Screening a panel of peptides with different acyl lysine modifications, we found that HDAC8 can catalyze the removal of acyl groups with 2-16 carbons from lysine 9 of the histone H3 peptide (H3K9). Interestingly, the catalytic efficiencies (k_cat/K_m) of HDAC8 on octanoyl, dodecanoyl, and myristoyl lysine are several-fold better than that on acetyl lysine. The increased catalytic efficiencies of HDAC8 on larger fatty acyl groups are due to the much lower K_m values. T-cell leukemia Jurkat cells treated with a HDAC8 specific inhibitor, PCI-34051, exhibited an increase in global fatty acylation compared to control treatment. Thus, the de-fatty-acylation activity of HDAC8 is likely physiologically relevant. This is the first report of a zinc-dependent HDAC with de-fatty-acylation activity, and identification of HDAC8 de-fatty-acylation targets will help to further understand the function of HDAC8 and protein lysine fatty acylation.

Figure 1

HPLC traces of HDAC8 activity assay with different acyl peptides as substrates. HDAC8 can remove the acetyl group, and other long chain acyl groups with different efficiencies.

Figure 2

HPLC traces showing HDAC8 selectivity on different histone peptides. (A-B) HDAC8 can catalyze deacetylation and demyristoylation on both H3K9 and H2BK12 peptides. (C) Only deacetylation activity was detected on H3K18 peptide. (D) No deacylation activity was detected on the H4K16 peptide.

Figure 3

Inhibition curves of PCI-34051 and SAHA on the demyristoylation and deacetylation activities of HDAC8.

Figure 4

Global fatty acylation Jurkat cells treated with PCI-34051. (A) Method for the detection of global protein lysine fatty acylation in Jurkat cells. Cells were cultured with the myristic acid analogue (Alk12) to allow metabolic labeling to occur. BODIPY-azide (B-N₃) was then conjugated to the alkyne group using click chemistry. The modifications on cysteine residues were removed by treating with 0.5 M hydroxylamine, pH 8.0, and the fluorescent signal was imaged after SDS-PAGE. (B) Chemical structures of different HDAC inhibitors. (C) Global fatty acylation of Jurkat cells treated with 50 μM PCI-34051 with different treatment times. The arrows point to several protein bands with elevated fatty acylation signals. (D) The increase in global fatty acylation was specific to PCI-34051. Jurkat cells treated with 25 μM of different HDAC inhibitors for 15 hrs. Only PCI-34051 treated cells showed higher fatty acylation when compared to the control and other HDAC inhibitor treated cells. (E) Western blot showing the global acetylation pattern of Jurkat cells treated with different HDAC inhibitors. SAHA and Parabinostat increased protein lysine acetylation while other inhibitors did not.